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(12) INTERNATIONAL APPLICATION PUBLISUfiD UND£K THE PATONT COOPERATION TREATY (PCT)

(19) World InteUectual Property /^^^^

IntemTotafBureau IliMnilllimilllllliilllllllllllllliillllllillim^

(43) International Publication Date (10) International Publication Number

10 September 2004 (10.09,2004) PCT WO 2004/077009 A2

(51) International Patent Classification'^:
(21) International Application Number:

GOIN

PCI7IL2004/00OI94

(22) International Filing Date: 26 February 2004 (26.02.2004)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:
154677
60/496,382
60/496,383
60/523,094
60/523,096

27 February 2003 (27.02.2003) IL

20 August 2003 (20.08.2003) US

19 November 2003 (19.11.2003) US

19 November 2003 (19.1 1 .2003) US

(71) Applicant (for all designated States except VS)i MOLEC-
ULAR CYTOMICS LTD. [CY/CY]; P.O. Box 21255,
Nicosia 1505 (CY).

(72) Inventor; and

(75) Inventor/Applicant (for US only): DEUTSCH,
Mordechai [IL/IL]; 73 Moshav Olesh, Doar Na
Lev-hasharon 42 855 (IL).

(74) Agent: G. E. EHRLICH (1995) LTD.; 1 1 Menachem Be-
gin Street, Ramat-gan 52 521 (IL).

(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, B A, BB, BG, BR, BW, BY, BZ, CA, CH, CN.
CO, CR, CU. CZ, DE, DK, DM. DZ, EC, EE, EG, ES. FI.
GB, GD, GE, GH, GM, HR, HU. ID, IL, IN, IS, JP, KE.
KG, KP, KR, KZ, LC, LK, LR, LS, LT. LU, LV, MA, MD,
MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
PH, PL, PT, RO, RU. SC, SD, SE, SG, SK, SL, SY, TJ, TM,
TN, TR, TT, TZ, UA, UG. US. UZ. VC, VN, YU, ZA, ZM.
ZW.

(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW. MZ, SD, SL, SZ, TZ, UG, ZM. ZW).
Eurasian (AM. AZ, BY. KG, KZ. MD, RU, TJ, TM). Euro-
pean (AT, BE, BG, CH. CY, CZ, DE, DK, EE, ES, H, FR,
GB, GR, HU, IE, rr, LU, MC, NL, PT, RO, SE, SI, SK,
TO), OAPl (BF, BJ, CF; CG, CI. CM, GA, GN. GQ, GW.
ML, MR, NE, SN, TD, TG).

Published:

— without international search report and to be republished
upon receipt of that report

For two -letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations" appearing at the begin-
ning of each regular issue of the PCT Gazette,

(54) TiUe: A METHOD AND DEVICE FOR MANIPULATING INDIVIDUAL SMALL OBJECTS

104

(57) Abstract: A method for individually moving small objects, such as cells, from one location to another as well as a device
for implementing the method is disclosed. A small object such as a cell is isolated at some initia] location and moved to some
destination location by the movement of the small object through a succession of intermediate locations until the small object arrives
at the destination location. Also disclosed are methods of manipulating cells made possible by the method and device of the present
1^ im^ention.

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A METHOD AND DEVICE FOR MANIPULATING INDIVIDUAL SMALL

OBJECTS

FIELD AND BACKGROUND OF THE INVENTION

5 The present invention relates to the field of micromanipulation and, more

particularly, to a method to and device for physically manipulating small objects,
especially living cells. The present invention also relates to the field of life sciences, and
more particularly to a device and methods for individually manipulating cells.

In the fields of biology and medicine it is often important to study the results of

10 the interaction between living cells and external factors such as environment, physical
stimuli, chemical stimuli and other cells. Such studies are important, for example, for
medical diagnostics, drug development and the understanding of basic cell function and
are generally divided into two types, static and dynamic studies.

In static studies a group of cells are exposed to whatever stimulus is being

15 studied and, after a certain time, the magnitude and nature of the reaction of the group
of cells to the stimulus eure determined.

In dynamic studies a single cell or a group of cells is exposed to whatever
stimulus is being studied and the magnitude and nature of the reaction of the cell or
group to the stimulus are determined, either continuously or discretely.

20 Both static and dynamic studies are often performed on a population of cells on

a macroscopic scale, in cuvettes or well arrays. One disadvantage of studying
populations of cells is that the results may teach of secondary effects rather than
primary effects of stimuli: the response of a cell to the response of other cells to a
stimulus is detected rather than the effect of the stimulus itself. Another disadvantage of

25 such studies is that the results reflect a distribution of effects on a group of cells. This
disadvantage is partially overcome by the use of homogenous populations of cells.
Although the study of homogenous populations of cells is informative, results are not
necessarily representative of "real-life" effects. Although theoretically possible, the
required manipulations using available technologies are too difficult and too slow when

30 applied to the study of individual cells.

Static and dynamic studies are also performed using flow cytometry. In flow
cytometry, the response of a cell to a stimulus is measured or detected for a single cells

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moving through a medium, the cell being discarded subsequently to the measxirement.
Flow-cytometry allows neither the study of an individual cell over a period of time nor
the study of the interaction of two or more specifically selected cells.

Practitioners of flow cytometry realize that many critical questions in cell
biology, developmental biology, inmiimology, oncology, pharmacology and virology
cannot be answered using even the most sophisticated flow cytometry techniques. It is
clear to one skilled in the art that complete understanding of complex biological
problem requires the dynamic manipulation and study of single cells or of a multiplicity
of cells studied as individuals in parallel or sequentially.

There is thus a widely recognized need for, and it would be highly advantageous
to have, a method to manipulate, isolate and study single, whole, living cells as
individuals.

SUMMARY OF THE INVENTION

The above needs are met by the present invention which allows isolation and
manipulation of an individual small object such as a single cell.

The present invention successfiiUy addresses the shortcomings of the prior art by
providing a method for moving a small object (such as a cell) from one location to
another location, thus allowing isolation and manipulation of the small object-^^jpie
present invention also provides a device for implementing the method of the present
invention.

According to the teachings of the present invention there is provided a method
of moving an individual small object from an initial location to a destination location
by:

a. defining a series of N+l locations Lq—Ln, N being an integer greater than 1
wherein Lq is the initial location and Ln is the destination location; and

b. for i from 0 to N-1, successively moving the individual small object from a
location Li to a location Li+i

wherein moving the small object is effected by applying an attractive force from
location Lj+i to the individual small object so as to cause the individual small object to
move from location Li to location Lj+i.

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According to an embodiment of the present invention, the position and/or
orientation of location Lj is fixed relative to the position and/or orientation of location
Li+i-

According to an embodiment of the present invention, to aid in moving the small
object, a repulsive force is applied to the individual small object from location L,- before,
during or after application of the attractive force from location Li+i.

According to a feature of the present invention, either or both an attractive force
or a repulsive force can be physical, electrical and/or magnetic.

According to a preferred embodiment of the present invention, a repulsive force
is a result of a flow of fluid from location Li, preferably the flow of fluid being
generated by injection of a fluid from the vicinity of location Li.

According to a preferred embodiment of the present invention, an attractive
force is a result of a flow of fluid towards location Lj+i, preferably the flow of fluid
being generated by suction of a fluid from the vicinity of location Lj+i .

According to a feature of the present invention, at least one (and preferably all)
of the locations Lo..,Lisi, includes an enclosure of a size sufficient to substantially
enclose (partially or completely) the individual small object (and preferably no more
than one small object at a time).

According to the teachings of the present invention there is also provided a
method of moving an individual small object in a fluid from a first location to a second
location by:

a. drawing fluid towards the first location so as to produce a first force localizing
the small object at the first location; and

b. subsequently drawing fluid towards the second location so as to produce a
second force localizing the small object at the second location.

According to an embodiment of the present invention, the position and/or
orientation of the first location is fixed relative to the position and/or orientation of the
second location.

According to an embodiment of the present invention, the drawing of fluid
towards the first location and the drawing of fluid towards the second location are
performed by suction of the fluid by a flow generator. Suitable flow generators include,
but aiS'not limited to, one or more pumps, and systems that use valves and the like.

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According to an embodiment of the present invention, before, diiring or after the
drawing of fluid towards the second location, the magnitude of the first force is reduced
so as to assist in locaUzing the small object at the second location.

According to an embodiment of the present invention, before, during or after the
drawing of fluid towards the second location, the first force is eliminated so as to assist
in localizing the small object at the second location.

According to an embodiment of the present iavention, before, during or after the
drawing of fluid towards the second location, fluid is injected from the first location so
as to produce a repulsive force to assist in localizing the small object at the second
location.

According to an embodiment of the present invention, the first location and the
second location include an enclosure of a size sufficient to substantially enclose
(partially or completely) the individual small object (and preferably no more than one
small object at a time). In such an embodiment, the drawing of fluids towards the first
5 location and towards the second location is preferably performed by suction of the
fluids from within a respective enclosure.

Depending on the specific embodiment of the present invention, the size of
individual small objects manipulated according to the teachings of the present invention
include objects is in the order of equal to or less than about 10^ micron, equal to or less
than about 10^ micron, equal to less than about 10* micron or equal to less than about
10^ micron. Such objects include crystals, bacteria, viruses, proteins, polymers,
macromolecules, ions and atoms, but especially cells.

In embodiments of the present invention using a fluid, it is preferred that the
fluid is a liquid, preferably a cell culture medium.

According to the teachings of the present invention there is also provided a cell
manipulation device, specifically configured to manipulate individual cells found in a
liquid such as a cell culture medium. The cell manipulation device according to the
teachings of the present invention includes:

a. at least N locations; and

b. at least two flow generators, each flow generator being fimctionally associated
with a location;

wherein N is at least two;

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who-ein at least two of the flow generators are independently settable to a mode
selected from the group of modes including a suction mode and an inactive mode;
and wherein at least two of the independently settable flow generators are each
associated with a different location. One embodiment of a device of the present
invention has two locations, each one of the two locations having a functionally
associated independently settable flow generator.

According to an embodiment of the present invention, the position and/or
orientation of at least one of the N locations is fixed relative to the position and/or
orientation of another one of the N locations. According to a preferred embodiment of
the present invention, all of the at least N locations having a fixed relative position
and/or orientation relative to the other locations.

According to an embodiment of the present invention, N is at least three and at
least three of the at least N locations are arranged in a one-dimensional array. By
one-dimensional array is meant that each one of the N locations is adjacent to no more
than two other locations.

According to an embodiment of the present invention, N is at least four and at
least four of the at least four locations are arranged in a two-dimensional array. By
two-dimensional array is meant that at least one of the N locations is adjacent to more
than two other locations.

According to an embodiment of the present invention, N is at least five and at
least five of the at least five locations are arranged in a rectangular lattice array. By
rectangular lattice array is meant that at least one of the N location is adjacent to four
other locations. Preferably, substantially all "non-border" locations are adjacent to foxir
other locations.

According to an embodiment of the present invention, N is at least seven and at
least seven of the at least seven locations are arranged in a hexagonal lattice array. By
hexagonal lattice array is meant that at least one of the N location is adjacent to six
other locations. Preferably, substantially all "non-border" locations are adjacent to six
other locations.

According to various embodiments of the present invention, N is at least ten, 19,
24, 36 and even more.

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According to an embodiment of the present invention, at least one of the flow
generators is independently settable to a mode selected from the group of modes
including a suction mode, an injection mode and an inactive mode. Preferably,
substantially all of the flow generators are independently settable to a mode selected
from the group of modes including a suction mode, an injection mode and an inactive
mode.

According to an embodiment of the present invention, at least one of the N
locations is associated with at least two flow generators and each one of these at least
two flow generators is independently settable to a mode selected from the group of
modes including a suction mode and an inactive mode (and preferably a suction mode,
an injection mode and an inactive mode).

According to an embodiment of the present invention, at least one of the N
locations is associated with at least three flow generators and each one of these at least
three flow generators is independently settable to a mode selected from the group of
modes including a suction mode and an inactive mode (and preferably a suction mode,
an injection mode and an inactive mode).

According to an embodiment of the present invention, at least two of the at least
N locations are substantially defined by the presence of an inlet of a respective
flow-generator.

According to an embodiment of the present invention, at least two of the at least
N locations are substantially depressions in a surface.

According to an embodiment of the present invention, at least two of the at least
N locations are substantially open trapping-ends of enclosures. Each such enclosure is
of a size sufficient to substantially enclose (partially or completely) a cell (and
preferably no more than one cell). According to a preferred embodiment, within at least
two of the at least two enclosures emerges an inlet of a respective flow generator. The
device of the present invention is specifically configured to manipulate cells.
Accordingly, the size of open trapping ends of enclosures is of a size sufficient to allow
entry of a cell to be studied, and preferably only one such cell at a time. Accordingly,
according to embodiments of the present invention, the order of the size of the open
trapping ends are equal to or less than about 10^ micron, equal to or less than about 10^

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micron, equal to or less than about 10^ micron and/or equal to or less than about 10®
micron.

According to an embodiment of the present invention, to allow for efficient
transfer of cells between one location and another location (in accordance with the
method of the present invention), at least one location is no more than 1000 micron
distant from at least one other location, preferably substantially every location being no
more than 1000 micron distant from at least one other location.

According to an embodiment of the present invention, to allow for efficient
transfer of cells between one location and another location (in accordance with the
method of the present invention), at least one location is no more than 100 micron
distant from at least one other location, preferably substantially every location being no
more than 100 micron distant from at least one other location.

According to an embodiment of the present invention, to allow for efficient
transfer of cells between one location and another location (in accordance with the
method of the present invention), at least one location is no more than 10 micron distant
from at least one other location,, preferably substantially every location being no more
than 10 micron distant from at least one other location.

According to an embodiment of the present invention, to allow for efficient
transfer of cells between one location and another location (in accordance with the
method of the present invention), at least one location is no more than 1 micron distant
from at least one other location, preferably substantially every location being no more
than 1 naicron distant from at least one other location.

According to an embodiment of the present invention, at least one of the
locations is configured so as to suspend a cell at distance above itself using a Venturi
effect, as explained hereinbelow in detail.

According to an embodiment of the present invention, at least one of the
locations is provided with a cell wall penetrating tool. According to a feature of the
present invention, each such cell wall penetrating tool is a member (including but not
limited to needles, wires and sharpened protuberances) pointing in a direction
substantially opposite a flow generated by a flow generator fiinctionally associated with
the location when set to a suction mode. As will be described hereinbelow in detail,

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when the flow generator is set to a suction mode, a cell is pulled towards and onto the
cell wall penetrating tool with sufficient force to effect cell wall penetration.

According to the teachings of the present invention there is also provided a
method of studying a cell by suspending the cell individually in a liquid by using a
Venturi effect. .

According to the teachings of the present invention there is provided a device for
suspending a cell in a liquid including: a. a substantially hollow body having an open
trapping-end and a central axis; b. emerging within the hollow body, at least one outlet
of a flow generator configured to inject liquid into the hollow body and through the
open trapping-end in a first flow substantially parallel to the central axis of the hollow
body; and c. emerging within the hollow body, at least one inlet of a flow generator
configured to draw liquid through the open trapping-end and out of the hollow body in a
second flow substantially parallel to the central axis of the hollow body; wherein the
first flow is closer to the central axis than the second flow. Such a cell suspension
device can be integrated into the cell manipulation device of the present invention,
where the hollow body defines, in part, a location or enclosure as described
hereinabove.

According to the teachings of the present invention there is also provided a
method of penetrating a cell wall by: a. immersing, in a liquid, a cell wall penetrating
tool pointing in a first direction; and b. applying suction from a second direction
opposite the first direction so as to cause a cell in the liquid to be carried against the cell
wall penetrating tool where the intensity of the carrying is sufficient to cause a wall of
the cell to be penetrated.

According to the teachings of the present invention there is also provided a
device for penetrating a cell wall including a. a substantially hollow body having an
open trapping-end; b. emerging within the hollow body, at least one inlet of a flow
generator configured to draw liquid out of the hollow body in a flow having a first
direction; and c. a cell wall penetrating tool pointing in a second direction substantially
opposite the first direction. Such a cell wall penetrating device can be integrated into fee
cell manipulation device of the present invention, where the hollow body defines, in
part, a location or enclosure as described hereinabove.

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According to the teachings of the present invention there is also provided a
method for selecting cells by: a. providing a group of cells; b. isolating cells from the
group of cells in an individual enclosure; c, examining each isolated cell; and d.
selecting isolated cells fulfilling at least one criterion. According to a feature of the
present invention, the selected cells are separated from the group of cells.

According to the teachings of the present invention there is also provided a
method for treating a cell-containing biological fluid by a. providing the cell-containing
biological fluid; b. identifying cells to be treated in the biological fluid; c. differentiating
between cells to be treated and other cells; and d. either or both:

i. physically separating the cells to be treated from the other cells; and / or

ii. directly manipulating the cells to be treated
thus treating the biological fluid.

According to a feature of the present invention, during the identifying, each cell
is isolated in an individual enclosure.

Examples of direct manipulation of the cells to be treated includes but is not
limited to destroying the cells to be treated (e.g,, physically, chemically, irradiation),
exposing the cells to be treated to a reagent {e.g. internally or externally, chemical
reagents, biological reagents), exposing the cells to be treated to radiative energy (e.g.
ultraviolet, infrared, visible light, coherent light, incoherent light, microwaves) and
penetrating the cell walls of the cells to be treated {e.g. to inoculate the cells or to
remove a sample from therewithin).

Examples of treatable biological liquids include but are not limited to lymphatic
fluids, blood, cerebral spinal fluids, semen, saliva, synovial fluid, bone marrow,
cochlear fluid, fluid extracted from tumors, ovarian fluid, amniotic fluid and chorionic
fluid.

According to an embodiment of the present invention, subsequent to d, the
biological liquid is directed into a living organism (both human and non-human).

According to an embodiment of the present invention, providing the biological
liquid includes a step of taking the biological liquid from a living organism (both himian
and non-human).

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According to an embodiment of the present invention, e. subsequently to d, the
biological liquid is directed into a Uving organism (both human and non-human) and
providing the biological liquid includes a step of directing the biological liquid to flow
from the living organism to the individual enclosures.

Unless otherwise defined, aU technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art. Although
methods and materials similar or eqiiivalent to those described herein can be used in the
practice or testing of the present invention, suitable methods and materials are described
5 below. All publications, patent applications, patents and other references mentioned
herein are incorporated by reference in their entirety. In case of conflict, the patent
specification, including definitions, will control. In addition, the materials, methods,
embodiments and examples are illustrative only and not intended to be limiting.

Implementation of the method and system of the present invention involves

10 performing or completing selected tasks or steps manually, automatically, or a
combination thereof. Moreover, according to actual instrumentation and equipment of
preferred embodiments of the method and system of the present invention, several
selected steps could be implemented by hardware or by software on any operating
system of any firmware or a combination thereof. For example, as hardware, selected

15 steps of the invention could be implemented as a chip or a circuit. As software, selected
steps of the invention could be implemented as a multiplicity of software instructions
being executed by a computer iising any suitable operating system. In any case, selected
steps of the method and system of the invention could be described as being performed
by a data processor, such as a computing platform for executing a multiplicity of

20 instructions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the
accompanying drawings. With specific reference now to the drawings in detail, it is
25 stressed that the particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present invention only, and

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are presented in the cause of providing what is believed to be the most useful and
readily understood description of the principles and conceptual aspects of the invention.
In this regard, no attempt is made to show structural details of the invention in more
detail than is necessary for a fundamental understanding of the invention, the
5 description taken with the drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
In the drawings:

FIGS. 1 A - IC illustrate the method of the present invention for moving a small
object from one location to a neighboring location using only attractive forces;
1 0 FIGS. 2 A - 2C illustrate the method of the present invention for moving a small

object from one location to a neighboring location using attractive and repulsive forces;

FIG. 3 illustrates the method of the present invention for moving a small object
from one location to a non-neighboring location by a series of successive transfers to a
series of neighboring locations;
15 FIG. 4 is a schematic depiction of a location of the present invention

implemented with a trapping enclosure;

FIG. 5 is a schematic depiction of a hexagonal matrix of locations of the present
invention;

FIG. 6A is a schematic depiction of a preferred embodiment of the cell
20 manipulation device of the present invention;

FIGS. 6B and 6C are schematic depictions of the tip of a preferred embodiment
of a probe tip end of a cell manipulation probe of the present invention;

FIG. 6D is a schematic depiction of an alternative embodiment of a probe tip
end of a cell manipulation probe;
25 FIG. 7A is a cross section view of a schematic depiction of a preferred

embodiment of a cell manipulation element with a single conduit;

FIG. 7B is a cross section view of a schematic depiction of a preferred
embodiment of a cell manipulation element with a single conduit and a cell stop;

FIG. 7C is a cross section view of a schematic depiction of a preferred
30 embodiment of a cell manipulation element with a single conduit and a bore narrowing;

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FIG. 7D is a cross section view of a schematic depiction of a preferred
embodiment of a cell manipulation element with an axial conduit and one peripheral
conduit;

FIG. 7E is a cross section view of a schem'atic depiction of a preferred
5 embodiment of a cell manipulation element with two peripheral conduits;

FIG. 7F is a diagram depicting a pump system useful for introducing reagents
through a cell manipulation element;

FIG. 7G is a cross section view of a schematic depiction of a preferred
embodiment of a cell manipulation element with a cell wall penetrating tool;
10 FIG. 7H is a cross section view of a schematic depiction of a preferred

embodiment of a cell manipulation element with an axial conduit equipped with a cell
wall penetrating tool;

FIG. 71 is a cross section view of a schematic depiction of a preferred
embodiment of a cell manipulation element with a retractable cell wall penetrating tool;
15 FIG. 8 A is a perspective view of a preferred embodiment of a cell manipulation

probe;

FIG. 8B is a cross section view of a schematic depiction of a preferred
embodiment of a cell manipxilation element;

FIGS. 9A-9C illustrate a method of producing a cell manipulation element from
20 glass tubes and rods;

FIGS. lOA-lOC are perspective views of modular cell manipulation elements;

FIG. lOD is a perspective view of an assembly plate used in conjunction with
modular cell manipulation elements to make a cell manipulation probe;

FIGS. 11 A- lie illustrate the transfer of a cell from one cell manipulation
25 element to a neighboring cell manipulation element;

FIG. 12 is a schematic depiction of the teachings of the present invention
applied to the on-line treatment of an organism;

FIG. 13 A is a perspective view illustrating the transfer of selected cells from a
cell manipulation probe to a biochip processor; and
30 FIG. 13B is a perspective view illustrating the transfer of selected cells from a

biochip processor to a cell manipulation probe.

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DETAILED DESCRIPTION OF THE INVENTION

The present invention is of a method and a device that allow the isolation and
manipulation of individual small objects, such as and especially living cells. With the
appropriate modifications, some embodiments of present invention allow the
5 practitioner to isolate, manipulate, observe and study an individual small object such as
a living- cell. By manipulate is meant to physically move but especially to expose to
many different and well-defined stimuli, including but not limited to chemical and
physical stimuli. Using the teachings of the present invention, complex multi-step
experiments can be reproducibly performed on single individual small objects such as
10 living cells.

The principles and operation of a device and method of the present invention
may be better understood with reference to the drawings and accompanying
descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be
15 understood that the device of the present invention is not limited in its application to the
details of construction and the arrangement of the components set forth in the following
description or illustrated in the drawings. It is also to be understood that the method
of the invention is not limited in its application to the details set forth in the following
description or exemplified by the specific embodiments. The invention is capable of
20 other embodiments or of being practiced or carried out in various ways. Also, it is to be
understood that the phraseology and terminology employed herein is for the purpose of
description and should not be regarded as limiting.

Method of Moving Small Obiects
25 A first aspect of the present invention is of a method for moving a small object

firom a first location to a second location, where preferably the relative position of the

locations to each other is fixed.

By small object is meant an object having dimensions in the order of less than

10^ microns, or even less than 10^ microns, or even less than 10^ microns or even less
30 than 10^ microns. Such objects include cells but also small particles, crystals, bacteria,

viruses, proteins, polymers, macromolecules and ions.

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The simplest embodiment of the method of the present invention, depicted in
Figures 1, involves:

a. applying a first attractive force 100 from a first location 102 on a small object
104, in order to localize small object 104 at first location 102 (Figure 1 A); and*

5 b. thereafter applying a second attractive force 106 firom a second location 108

on small object 104 (Figure IB).

As is clear to one skilled in the art, in such a way small object 104 is transferred
from first location 102 to second location 108. As is also clear to one skilled in the art,
the proximity and relative spatial orientation of first location 102 and second location

10 108 must be selected so as to allow efficient transfer of small object 104.

To expedite the transfer of small object 104, the intensity of first attractive force
100 is preferably reduced or eliminated. Reduction or elimination of first attractive
force 100 occurs before, during or after application of second attractive force 106. In
some embodiments of the method of the present invention the first attractive force is not

15 only reduced but also reversed to be a repulsive force. In such a case, the small object is
sequentially or simultaneously repelled from first location 102 and attracted to second
location 108, Figures 2.

The method of the present invention is preferably applied to the moving of some
small object from some initial location to some destination location by the movement of

20 the small object through a succession of intemiediate locations until the small object
arrives at the destination location. Preferably, the position of the locations relative to
each other is fixed. This process is schematically depicted in Figure 3 for the movement
of small object 104 from an initial location 110 to a destination location 114 by the
movement of small object 104 from initial location 110 througji a succession of three

25 intermediate locations 112a, 112b and 112c until small object 104 arrives at destination
location 114. The movement of small object 104 between any two locations is
analogous to the movement between first location 102 and second location 108 as
depicted in Figures 1 and Figures 2 discussed hereinabove.

When the method of the present invention is applied to a system where there are

30 more than two locations then any two locations are related to each other in one of two
ways: neighboring and not-neighboring. Neighboring locations are those locations
where to a small object can be directly transferred in one step. Non-neighboring

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locations are those locations where to a small object can be transferred only via other
locations. In Figure 3, location 110 and location 112b are neighboring to location 112a.
However location 110 is not-neigjiboring to location 112b.

The physical nature of the different locations as well as the nature of the'

5 respective attractive and, if implemented, repulsive forces depends on the size and .
nature of the small objects to be manipulated.

The attractive and repulsive forces can be physical, electrical, magnetic or any
combination thereof. Electric forces can include those made by charging a surface, but
can also be of chemical origin such as ionic bonds or hydrogen bonds. Preferably the

10 attractive and repulsive forces eire implemented by a flow of a physical medium such as
a gas or a liquid. When an attractive force is generated by the flow of a physical
medium, then the attractive force associated with a location is generally implemented by
suction of a fluid through or in the immediate vicinity of that location. When a repulsive
force is generated by the flow of a physical medium, then the repulsive force associated

15 with a location is generally implemented by injection of a fluid through or in the
immediate vicinity of that location.

As stated above, the physical nature of the locations is determined by the size
and nature of the small object to be manipulated and the nature of the forces applied to
manipulate the object. In some embodiments of the present invention, the locations are

20 simply areas, for example, on a surface, defined by the proxinnity of a respective
attractive force. In other embodiments, the designated locations are dimples or recesses
on a surface.

In preferred embodiments of the present invention, especially suited for the
manipulation of cells, the locations are trapping enclosures (e.g., chambers or tubes)

25 configured to substantially physically contain (partially or completely) a small object,
such as a cell. In such preferred embodiments, schematically depicted in Figure 4 in
cross section, a trapping enclosure 116 is provided with an open trapping-end 118
through which a respective attractive or repulsive force is applied and through which a
manipulated small object 120 enters and exits trapping enclosure 116. It is preferred that

30 the dimensions of open trapping-end 118 are such that only one small object 120 can
enter trapping enclosure 116 at one time and that once a first small object 120 enters
any other small object, e,g, 122, is blocked firom entering trapping enclosure 116. The

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geometry of open trapping-end 118 is not generally of significance and can be fashioned
in any shape, for example, square, circular or hexagonal.

Cells are ordinarily found in a liquid medium. Thus, for embodiments of the
present invention directed to the manipulation of cells, an attractive force is preferably
5 implemented using a flow generator 124 operated in a suction mode. Analogously, in
such embodiments a repulsive force is preferably implemented using a flow generator
124 operated in an injection mode. The inlet / outlet of flow generator 124 is preferably
attached to trapping enclosure 116 in a way so that attractive and repulsive forces act
through open trapping-end 118. hi Figure 4, the inlet / outlet of flow generator 124 is
10 attached to trapping enclosure 116 through conduit 126 which emerges inside trapping
enclosure 116.

By a flow generator is meant a device or other means to generate a flow with a
desired direction in a fluid. An example of a flow generator is a pump or plurality of
pumps. In some embodiments a flow generator include valves and the like.

15 For embodiments of the present invention directed to the manipulation of cells,

the fluid preferably used is a liquid media, especially a cell culture media. Many
different cell culture media are known to one skilled in the art. Examples of cell culture
media that are useful in implementing the teachings of the present invention include but
are not limited to Dulbecco*s minimal essential medium, Hank's medium and those

20 listed in U.S. Patent 6,692,961, U.S. Patent 6,689,594, U.S. Patent 6,686,190, U.S.
Patent 6,673,591, U.S. Patent 6,670,184, U.S. Patent 6,670,180, U.S. Patent 6,667,034,
U.S. Patent 6,660,501, U.S. Patent 6,649,408, U.S. Patent 6,642,050, U.S. Patent
6,635,448, U.S. Patent 6,627,426, U.S. Patent 6,610,516, U.S. Patent 6,593,140, U.S.
Patent 6,589,765, U.S. Patent 6,588,586, U.S. Patent 6,569,422, U.S. Patent 6,555,365,

25 U.S. Patent 6,544,788, U.S. Patent 6,528,286, U.S. Patent 6,511,430, U.S. Patent
6,506,598, U.S. Patent 6,492,163, U.S. Patent 6,492,148, U.S. Patent 6,489,144, U.S.
Patent 6,479,252, U.S. Patent 6,468,788, U.S. Patent 6,465,205, U.S. Patent 6,465,000,
U.S. Patent 6,455,310, U.S. Patent 6,413,746, U.S. Patent 6,413,744, U.S. Patent
6,410,309, U.S. Patent 6,403,369, U.S. Patent 6,383,810, U.S. Patent 6,378,527, U.S.

30 Patent 6,372,494 , U.S. Patent 6,344,354, U.S. Patent 6,342,384, U.S. Patent 6,338,964,
U.S. Patent 6,333,192 and U.S. Patent 6,329,195.

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When manipulating a small object according to the present invention it is often
advantageous to hold the small object at a given location for a desired length of time.
Holding a small object at a given location can be implemented in many different ways.
One preferred method of holding a small object at a given location is by the continuous
application of an associated attractive force. In Figure 4, such a method is implemented
by continuously activating flow generator 124 in suction mode. The use of such a
method of holding the small object in place is generally contingent on the presence of a
"stop", that is a physical impediment to small object 120 being sucked into flow
generator 124 by the attractive force. In Figure 4 the stop is substantially the small
dimension, relative to the size of small object 120, of conduit 126.

Another preferred method of holding a small object at a given location is by
relying on the nature of the small object and the geometry of the location to prevent
drifting of the small object away from the location. In Figure 4, such a method is
implemented by setting flow generator 124 to inactive mode, a mode where neither an
attractive nor a repulsive force is applied. Due to the small size of open trapping-end
118 and the lack of currents inside trapping enclosure 116, small object 120 remains
trapped in the location defined by trapping enclosure 116.

In one embodiment of the present invention, locations are disposed in a
one-dimensional array, understood to mean that any location has at most two
neighboring locations. Examples of one-dimensional arrays include linear arrangements
of locations, curved arrangements of locations and circular arrangements of locations.

In another embodiment of the present invention, locations are disposed in a
two-dimensional array, understood to mean that at least one given location has more
than two neighboring locations. Two-dimensional arrays include Y-shaped and
X-shaped arrays. Preferably a two-dimensional array of the present invention is a matrix
arrangement, that is a regular arrangement of a multiplicity of locations. One preferred
matrix arrangement of locations is a square lattice, where substantially each non-edge
location has four neighboring locations. Another preferred matrix arrangement of
locations is a hexagonal lattice, where substantially each non-edge location has six
neighboring locations. In Figure 5 a hexagonal matrix of locations 128 is depicted, each
location 128 having a circular cross section.

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It is important to note that for the sake of simpUcity, arrays of locations are
depicted and described herein as being substantially planar, that is, the locations or the
open trapping-ends of the locations as a group substantially define a plane. This is done
exclusively for the sake of simplicity of explanation. In some embodiments of the
5 present invention a matrix of locations implementing the method of the present
invention may be non-planar.

Cell Manipulation Device

For the manipulation of cells the method of the present invention is preferably
10 implemented using a cell-manipulation device of the present invention, the
cell-manipulation device including at least two (but preferably a multiplicity) of
cell-manipulation elements of the present invention, the at least two (or the multiplicity)
of cell-manipulation elements defining a cell-manipulation probe of the present
invention.

15 A preferred embodiment and the currently known best mode of implementing

the device of the present invention is described hereinbelow and with reference, inter
alia, to Figures 6. It is understood that cell-manipulation device 130 depicted in Figures
6 and described hereinbelow is a particular specific device suitable for manipulating
cells in a liquid medium, and described herein exclusively for exemplary purposes.

20 The central component of device 130, a cell-manipulation probe 132 having a

probe-end 134 and a butt end 136 is depicted in Figure 6A. Surrounding probe-end 134
is vial 138. Cell manipulation probe 132 is made up of a multiplicity of bundled
cell-manipulation elements. Functionally associated with each cell-manipulation
element is one, two, three or even more independently controllable pumps 142. Each

25 pump 142 is connected to a respective cell-manipulation element through butt end 136
of cell manipulation probe 132. For clarity, only a limited number of pumps 142 and
connections to cell-manipulation elements are depicted in Figure 6A. Controlling the
activation of pumps 142 is central control unit 144^ substantially a programmable
computer with necessary hardware and software to receive commands from a user input

30 interface 146, to perform the received commands, for example by performing necessary
calculations or activating a specific pump 142, and to display information using a user
output interface 148. Device 130 is also provided with an observation component 150,

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such as but not limited to a CCD camera equipped with a microscope lens. Observation
component 150 in Figure 6A is functionally associated with central control unit 144.
Observation component 150 receives commands from central control unit 144 and
returns data corresponding to acquired images, especially of cells manipulated at
5 probe-end 134 of cell manipulation probe 132. Central control unit 144 is configured to
display data received from observation component 150 and display the data in user
understandable form through user output interface 148. It is preferred that central
control unit 144 be configured with image processing capabilities so as to "interpret"
video data received from observation component 150 and react to the interpreted data.

10 In Figure 6B, a close-up of probe-end 134 of cell-manipulation probe 132

without vial 138 is depicted. A multiplicity of hexagonal trapping enclosures 140, each
being a part of a cell-manipulation element, are depicted with a cell 142 trapped therein.
Trapping enclosures 140 are arrayed in a matrix 145. A semi-circular isolation inlet 146
of an isolation element and a semi-circular waste inlet 148 of a waste element are

15 positioned at the circumference of matrix 145. In the center of matrix 145 is a multi-cell
reaction enclosure 151 of a multi-cell reaction element.

In Figure 6C, probe-end 134 of cell manipulation probe 132 with vial 138 is
depicted close-up. Emerging into vial 138 are sample inlet 152, fluid inlet 154 and vial
reagent inlet 156. Each such inlet is functionally associated with a respective pump 158,

20 160 and 162 to allow introduction of samples, fluid or reagents, respectively, onto
probe-end 134. Pumps 158, 160 and 162 are independently controllable by central
control unit 144.

Cell'Manipidation Elements
25 As stated hereinabove, cell-manipulation probe 132 is substantially made up of a

multiplicity of individual cell-manipulation elements. There are many variations of
cell-manipulation elements, some of which are discussed hereinbelow.

Types of cell-manipulation elements
30 One simple cell manipulation element preferred in implementing a device of the

present invention is a dimple on a surface, where at the bottom of the dimple is the inlet

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of an functionally associated flow generator having two modes: a suction mode (to
•generate an attractive force) and an inactive mode. When the flow generator is set to
suction mode, a cell is attracted and held at the associated dimple by the produced
suction. When the pump is set to inactive mode, the held cell may drift away. If a pump

5 functionally associated with a neighboring dimple is set to suction mode, the cell drifts
towards and is held in that neighboring dimple in accordance with the method of the
present invention. Although such a cell manipulation element is simple to produce and
operate, and a cell-manipulation probe made of an array of such cell manipulation
elements effectively implements the method of the present invention, the utility of such

10 a cell-manipulation probe is limited as it is difficult to expose only selected cells to
stimuli such as reagents, vide infra.

For embodiments of the present invention directed to the manipulation of cells,
it is generally preferred that a cell manipulation element have a trapping enclosure with
an open trapping end as depicted in Figure 4. The trapping enclosure is configured so as

15 to substantially physically contain (partially or completely) a cell held therein while the
open trapping end provides access of cells into and out of the trapping enclosure. In
such a way a cell in the trapping enclosure is isolated, allowing selective exposure of
cells to stimuli such as reagents. The inlet / outlet of a flow generator associated with a
cell manipulation element having a trapping enclosure advantageously emerges within

20 the trapping enclosure. In some embodiments such a flow generator has only two
modes: a suction mode and an inactive mode. In a preferred embodiment, such a
trapping enclosure is configured or shaped so that when the flow generator applies no
force {e.g. is set to inactive mode), a held cell is retained within the trapping enclosure.
When the trapping enclosure is so configures, an associated flow generator preferably

25 has at least three modes: a suction mode (to generate an attractive force), an injection
mode (to generate a repulsive force) and an inactive mode. When the flow generator is
set to suction mode, fluid is drawn firom a trapping enclosure through a respective
conduit generating an attractive force to pull a nearby cell into the trapping enclosure.
When the flow generator is set to inactive mode, a held cell remains within the trapping

30 enclosure. When the flow generator is set to injection mode, fluid is injected into a
trapping enclosure through a respective conduit generating a repulsive force to eject a
held cell firom the trapping enclosure.

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It is generally preferred that a trapping enclosure and an open trapping end of a
cell manipulation element have dimensions close to those of the cell to be held and
manipulated. As is known to one skilled in the art, cells of various sizes exist. Plant
cells are typically large, having a diameter of between 10^ to 10^ microns. Animal cells
5 typically have a diameter of between 10*^ to 10^ microns. Bacteria are typically small,
having a diameter of between 10'^ to 10'^ microns. It is therefore preferred that the
dimensions of the open trapping-ends of the trapping enclosure be determined by the
size of cells that are to be studied with a specific device of the present invention.

10 Cell manipulation elements associated with a single conduit pump

A simple cell manipulation element 164 implementing a trapping enclosure is
schematically depicted in Figure 7A. Cell manipulation element 164 is substantially a
tube 166 functionally associated with a conduit pump (not depicted), the conduit pump
operable in a suction mode, an injection mode and an inactive mode. In

15 cell-manipulation element 164 the end of tube 166 serves as an open trapping-end 170
and the bore of tube 166 serves as a trapping enclosure 140. The bore of tube 166 also
serves as a conduit 174 for transporting fluid from the functionally associated conduit
pump to attract and repel a cell in accordance with the teachings of the present
invention.

20 It is often preferred that a cell manipulation element have a cell stop, a physical

obstruction limiting the extent of penetration of a cell inside a cell manipulation
element. Depending on the specific embodiment, a cell stop is a narrowing, a protrusion
or other physical structural feature that limits the distance into an functionally
associated trapping enclosure that a cell can be drawn,

25 A preferred cell manipulation element 176 integrating a cell stop is depicted in

Figure 7B. Cell manipulation element 176 is similar to cell manipulation element 164
except that cell manipulation element 176 is also provided with a cell stop 178. Cell
stop 178 obstructs the bore of tube 166 so that a cell entering through open trapping-end
170 penetrates until making contact with cell stop 178. Trapping enclosure 140 of cell

30 manipulation element 176 is defined by the inside surface of tube 166 and cell stop 178.
The volume between cell stop 178 and tube 166 serves as a single conduit 174 for
transporting fluid from a functionally associated conduit pump to attract and repel a cell

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in accordance with the teachings of the present invention. It is inniportant to note that in
some embodiments conduit 174 has an annular cross section. In other embodiments
conduit 174 is made of a multipHcity of individual fluid paths peripherally arrayed
about cell stop 178. In such an embodiment, all the individual fluid paths preferably

5 transport fluid from a single conduit pump.

An additional cell manipulation element 180 integrating a cell stop is depicted in
Figure 7C. In cell manipulation element 180 the function of a cell stop is performed by
a narrowing 181 of the bore of tube 166 to an extent that a cell entering through open
trapping-end 170 cannot penetrate beyond naixowing 181. Thus, trapping enclosure 140

10 of cell manipulation element 180 is defined by the inside surface of tube 166 and
narrowing 181 in the bore of tube 166. As in cell manipulation element 164, the bore of
tube 166 serves as a conduit 174 for fluid from a ftmctionally associated conduit pump.

Cell manipulation elements 164, 176 and 180 are all operated in a substantially
similar way. Central control unit 144 sets a functionally associated conduit pump to

15 suction mode, producing an attractive force through conduit 174 to attract cells in the
vicinity of an open trapping-end 170 to enter cell manipulation element 164, 176 or 180
through an open trapping-end 170 and to be trapped in a trapping enclosure 140. When
cell manipulation element 164 is ixsed, central control unit 144 sets a functionally
associated conduit pump to inactive mode when a cell enters open trapping-end 170 to

20 prevent penetration of the cell too far into the bore of tube 166 and consequent loss of
the cell. It is thus generally preferred to use a cell manipulation element such as 164
under observation of an observation component 150. In contrast, a cell entering open
trapping-end 170 of a cell manipulation element 176 or 180 makes contact with a
functionally associated cell stop 178 or narrowing 181, respectively, and thus remains

25 held in trapping enclosure 140.

When it is desired to eject a cell from a trapping enclosure 140 of a cell
manipulation element 164, 176 or 180, central control unit 144 sets a respective
functionally associated conduit pump to injection mode. Fluid is forced through conduit
174 pushing a held cell outwards from trapping enclosure 140 through open trapping

30 end 170.

Cell manipulation elements functionally associated with multiple conduit pumps

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To increase the flexibility of the types of cell manipulations that can be
performed using a device of the present invention, it is often advantageous to use a cell
manipulation element having more than one conduit and functionally associated with
more than one independently controllable conduit pump.
5 Cell manipulation element 182, depicted in Figure 7D, has more than one

conduit and is functionally associated with more than one independently controllable
conduit pump. Cell manipulation element 182 is related to cell manipulation element
176 in that a trapping enclosure 140 is defined by the inside surface of a tube 166 and
by a cell stop 184. Further, the volume between tube 166 and cell stop 184 serves as a
10 first peripheral conduit 174 for fluid firom a functionally associated first conduit pump.
Cell manipulation element 182 is characterized, however, in that cell stop 184 is
substantially a hollow tube. The bore of cell stop 184 serves as a second axial fluid
conduit 186 transporting fluid from an independently controlled and activated second
conduit pump.

15 An additional preferred cell manipulation element functionally associated with

more than one independently controllable conduit pump is 188 depicted in Figure 7E.
Cell manipulation element 188 is related to cell manipulation element 176 in that a
trapping enclosure 140 is defined by the inside surface of a tube 166 and by a cell stop
178. However, in cell manipulation element 178, the volume between tube 166 and cell

20 stop 178 does not have an annular cross section but is rather made up of at least two
distinct conduits found at the periphery of tube 106, a first peripheral conduit 174 and a
second peripheral conduit 186. First peripheral conduit 174 is functionally associated
with a first conduit pump. Second peripheral conduit 186 is functionally associated with
an independently controlable second conduit pump.

25 It is important to note that variants of cell manipulation element 188 are

provided with more than two (e.g. three, four, five, six or even more) conduits
(especially peripheral conduits) each such conduit associated with an individually
controllable conduit pximp.

For loading a cell into a cell manipulation element fimctionally associated with

30 two independently controllable pumps, a fimctionally associated first conduit pump and
/ or second conduit pump are set to suction mode by central control unit 144. If only one
associated conduit pump is used, the other associated conduit pump is set to inactive

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mode. Analogously, ejection of a cell from trapping enclosure 140 of cell manipulation
element 182 is performed when a functionally associated first conduit pump and / or
second conduit pump are set to injection mode by central control unit 144. If only one
associated conduit pump is used, the other associated conduit pump is set to inactive
5 mode.

A useful function that can be implemented using a cell manipulation element
functionally associated with two or more independently controllable conduit pumps is
washing of a cell held in a respective trapping enclosure 140. For a cell manipulation
element 188, central control unit 144 sets one functionally associated conduit pump to

10 suction mode and the other functionally associated conduit pump to injection mode.
Liquid flows from one conduit to another, thus washing a cell held in trapping enclosure
140. For a cell manipulation element 182, central control unit 144 sets a first conduit
pump functionally associated with peripheral conduit 174 to injection mode and sets a
second conduit pump functionally associated with axial conduit 186 to suction mode.

15 Liquid flows from one conduit to the other, thus washing a cell held in trapping
enclosure 140.

A useful function that can be performed using cell manipulation element 182 is
suspending a cell held in trapping enclosure 140 at a distance from open trapping-end
170. To perform the suspension function, central control \mit 144 sets the conduit pump

20 functionally associated with axial conduit 186 to injection mode while setting the
conduit pump functionally associated with peripheral conduit 174 to suction mode. The
resulting Ventxiri effect suspends a cell originally trapped in trapping enclosure 140
above open trapping-end 170. The suspension function allows a cell to be temporarily
removed from trapping enclosure 140 to allow clear, unimpeded examination of the cell

25 from many directions, a challenging task when a cell is contained within trapping
enclosure 140. The suspension function as described hereinabove may be generally
performed with a properly configured cell manipulation element having at least one
axial conduit and at least one peripheral conduit. If there is only one peripheral conduit,
that peripheral conduit is preferably substantially annular. If there is more than one

30 peripheral conduit, the peripheral conduits are preferably arrayed symmetrically about
the axial conduit.

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An additional useful function that can be implemented using a cell manipulation
element functionally associated with two or more independently controllable conduit
pumps, such as 182 or 188, is selective exposure of a cell held in trapping enclosure 140
to a reagent. The function of selectively exposing a cell to a reagent will be discussed
S herein with reference to a cell manipulation element 188. In one embodiment, exposure
is performed by providing one of the two conduit pumps functionally associated with
cell manipulation element 188 with a reagent. When it is desired to selectively expose a
cell to the reagent, a process analogous to washing a cell as described above is foUowed.
The associated conduit pump provided with reagent is set to injection mode bringing

10 reagent into trapping enclosure 140 and thus to contact a cell held therein. In parallel the
second associated conduit pump is set to suction mode, drawing away reagent and
preventing leakage of reagent out through a open trapping-end 170. A disadvantage of
such an implementation is that a conduit pump provided with reagent cannot be used for
performing other functions.

15 Selective exposure of a cell can also be achieved in a cell manipulation element

provided with three conduits each functionally associated with an independently
controllable conduit pump, where one conduit is dedicated to providing reagent and the
other two conduits only for performing other functions.

In a preferred embodiment for implementing selective exposure of cells to a

20 reagent, a cell manipulation element is functionally associated with three pumps as
depicted in Figure 7F. In Figure 7F is depicted a cell manipulation element 188 where
conduit 174 is functionally associated with a first conduit pump 190 and conduit 186 is
functionally associated with a second conduit pump 192. On the fluid-line cormecting
conduit 174 with first conduit pump 190 is reagent injection pump 194. Similarly, to the

25 cell washing function discussed hereinabove, when it is desired to expose a cell to a
reagent, central control unit 144 sets first conduit pump 190 to injection mode and
second conduit pump 192 to suction mode. Further, central control unit 144 sets reagent
injection pump 194 to injection mode, causing reagent fi-om a container 196 to enter the
flow of first conduit pump 190 towards conduit 174. The reagent is transported througji

30 conduit 174, exposing a cell held in trapping enclosure 140 to the reagent Reagent is
then pumped away through conduit 186 by the action of second conduit pump 192,

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drawing away reagent and preventing leakage of reagent out through a open
trapping-end 170.

Cell manipulation elements having a tool for penetrating a cell wall
5 .It is often advantageous to be able to introduce one or more reagents directly

into a cell or to extract a sample from inside a cell. To this end, in a preferred
embodiment of the present invention, a cell manipulation element is provided with a
tool configured to penetrate a cell wall. Such a tool is, for example, a pointed solid
object, for example from glass or metal. Preferably, such a tool is disposed inside the

10 trapping enclosure of the cell manipulation element in such a way that when an
attractive force according to the present invention is applied, the cell is pulled against
the tool and the cell wall consequendy penetrated.

In Figure 7G a cell manipulation element 198 analogous to cell manipulation
element 188 is depicted, having a cell wall penetrating tool 200. Cell wall penetrating

15 tool 200 is a stainless steel wire inside cell stop 178. To penetrate the wall of a cell, the
cell is brought into trapping enclosure 140. Conduit pumps functionally associated with
conduits 174 and 186 are set to suction mode. The resulting attractive force pulls the
cell onto cell wall penetrating tool 200, thus penetrating the cell wall. Post-penetrating
cell release is accomplished by setting the fiinctionally associated conduit pumps to

20 injection mode so that the resulting liquid flow through conduits 174 and 186 pushes the
penetrated cell off of cell wall penetrating tool 200.

Advantageous is that once a cell wall is penetrated, a reagent is injected into the
penetrated cell or the contents of the penetrated cell are sampled. In a preferred
embodiment that a cell wall penetrating tool has a conduit functionally associated with

25 an independently controllable pimip for injecting reagents or sampling cell contents
through the conduit.

In Figure 7H a cell manipulation element 202 analogous to cell manipulation
element 182 is depicted, having a cell wall penetrating tool 204. Cell wall penetrating
tool 204 is substantially a sharpened tip of hollow cell stop 184. To penetrate the wall of
30 a cell, the cell is brought into trapping enclosure 140. The conduit pumps functionally
associated with peripheral conduit 174 are set to suction mode so that the resulting
Hquid flow pulls the cell onto cell wall penetrating tool 204, penetrating tihe cell wall. If

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it is desired to extract a sample from inside the cell, the conduit pump functionally
associated with axial conduit 186 is set to suction mode. If it is desired to inject a
reagent into the cell, the conduit pump functionally associated with axial conduit 186 is
set to injection niode. As described for cell manipulation element 198, post-penetrating
cell release is accomplished by setting conduit pumps functionally associated with
peripheral conduit 174 to injection mode so that the resulting liquid flow pushes the
penetrated cell off from cell wall penetrating tool 204.

An alternative method for releasing a penetrated cell from a cell wall penetrating
tool is by providing a cell manipulation element 206 with a retractable cell wall
penetrating tool 208 as depicted in Figure 71. Cell manipulation element 206 is similar
to cell manipulation element 182 excepting that a cell wall penetrating tool 208, a
stainless steel wire, is threaded through bore I86.T0 penetrate the wall of a cell, the cell
is brought into trapping enclosure 140. Pumps functionally associated with conduits 174
and 186 are set to suction mode. The resulting attractive force pulls the cell onto cell
wall penetrating tool 208, thus penetrating the cell wall. Post-penetrating cell release is
accomplished by pulling cell wall penetrating tool 208 outwards from the penetrated
cell through bore 186.

Multi-cell reaction element

A multi-cell reaction element, of which only a multi-cell reaction enclosure 150
is depicted in Figure 6B, is a substantially a cell-manipulation element having an open
trapping-end and a multi-cell reaction enclosure 150 both large enough to accommodate
at least two cells rather than only one cell like a standard cell-manipulation element as
described hereinabove. A multi-cell reaction element can be substantially of any of the
types of elements discussed hereinabove (especially as related to the number and
arrangement of conduits) and is functionally associated with at least one independently
controllable conduit pump.

A multi-cell reaction element provides a venue to isolate two or more cells in
one place to enable interaction of the two or more cells. Operation of a multi-cell
reaction element is analogous to the operation of cell manipulation elements as
described above. As depicted in Figure 6B, at least two cells 142, each isolated in a
separate trapping enclosure 140 are moved according to the method of the present

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invention to trapping enclosures 140 neighboring mnlti-cell reaction enclosure 150. The
at least two cells 142 are expelled from respective trapping enclosures 140 while a
conduit pump functionally associated with the multi-cell reaction element is set to
suction mode. The resulting forces draw the expelled cells into multi-cell reaction
5 enclosure 150. Inside multi-cell reaction enclosure 150 the at least two cells 142
interact. If desired, reagents are added as described hereinabove and hereinbelow. When
the cell interaction is completed the at least two cells 142 are expelled from multi-cell
reaction enclosure 150. The at least two cells 142 can be discarded or reisolated in
trapping enclosures 140 in the usual way.
10 Importantly, in a preferred embodiment a cell manipulation probe of the present

invention is provided with more than one multi-cell reaction element.

Sample introduction element

In Figure 6D, in the center of matrix 145 emerges the outlet of sample
15 introduction element 163. Sample introduction element is substantially a tube
functionally associated with an inlet pump (not depicted). Samples containing cells that
are to be manipulated using the device of the present invention are introduced through
sample introduction element 163.

20 Isolation element

An isolation element, of which only a substantially semi-circular isolation inlet
146 is depicted in Figure 6B, is substantially a multi-cell reaction element with
significant structural differences arising from the different uses for which these two
elements are intended. An isolation element is primarily intended to provide a location

25 where a user of a device of the present invention concentrates a relatively large number
of cells without necessarily intending to later manipulate the cells as individuals. For
example, certain cells can be sorted from a large group of cells, and only the certain
cells are selected and transferred to an isolation enclosure of the isolation element From
the isolation enclosure the certain cells are removed, either in accordance with the

30 method of the present invention or in some other manner. Therefore, an isolation
enclosure that is part of an isolation element is generally large enough to accommodate
a relatively large volume of fluid and many cells. At a minimvim, isolation element is

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functionally associated with an isolation pump configured to operate in a suction mode.
When the isolation pump is operated, cells are drawn into the isolation enclosure of the
isolation element. In some embodiments, an isolation element is functionally associated
with an isolation pump configured to operate also in an injection mode. When the
5 isolation pump is operated in injection mode, cells trapped in the isolation enclosure are
expelled back to the vicinity of probe tip 134. Preferably, the isolation enclosure of an
isolation element is provided with a diaphragm or other valve-like structural feature. A
user can, using a needle-equipped syringe (or other appropriate device), draw out cells
and fluids isolated in the isolation enclosure.
10 hnportantly, in a preferred embodiment a cell manipulation probe of the present

invention is provided with more than one isolation element.

Waste element

A waste element, of which only a substantially semi-circular waste inlet 148 is
1 5 depicted in Figure 6B, is substantially a tubular element functionally associated with a
waste pump operable in suction mode. The function of waste element is to remove
undesired or not needed substances from the proximity of probe-end 134 of
cell-manipulation probe 132, and especially firom matrix 145. When a fiinctionally
associated waste pump is activated, cells, fluid, cell debris and the like are drawn
20 through waste inlet 148 of the waste element and discarded.

Cell-Manipulation Probe

A cell-manipulation probe of the present invention is composed of at least two
but preferably a multiplicity of individual cell-manipulation elements as described

25 above. The cell-manipulation elements are arranged so that the open trapping-ends of
the cell-manipulation- elements are in functional proximity of each other, that is at a
distance and orientation so that cells can be transferred jfrom a first cell-manipulation
element to a neighboring cell-manipulation element according to the method of the
present invention. Preferably but not necessarily a cell-manipulation probe is also

30 provided with a sample introduction element, waste element, an isolation element
and/or a multi-cell reaction element.

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In some embodiments, a cell-manipulation probe may be homogenous, that is
that all the component cell-manipulation elements are substantially the same. In other
embodiments, a cell-manipulation probe is heterogeneous, that is the cell-manipulation
probe includes a variety of different cell-mandpulation elements. Differences between
5 the cell-manipulation elements making up a heterogeneous cell manipulation probe
potentially include the geometry of the trapping enclosures (especially size of the
trapping enclosure or size of the open trapping-ends), the number of conduits and the
arrangements of conduits. A cell-manipulation probe potentially includes some or all of
the types of cell manipulation elements discussed hereinabove, but potentially types of

10 cell-manipulation elements not discussed herein but may arise are also included. The
utility of a heterogeneous cell-manipulation probe is discussed hereinbelow.

In a preferred embodiment, a cell manipulation probe is a bundle of cell
manipulation elements. In Figure 8A a cell manipulation probe 132 is depicted. In
Figure 8B a component cell manipulation element 210 similar to cell manipulation

1 5 element 176 is depicted in cross section. As seen in Figure 6A and in Figure 8A a cell
manipulation probe 132 has a probe-end 134 and a butt end 136. As depicted in Figure
8A, generally speaking, at butt end 136 of cell manipulation probe 132 the ends of the
individual cell manipulation elements 210 are relatively large (~10^ microns, 1000 to
2000 microns) so as to allow simple maintenance and attachment of conduits and cell

20 manipulation elements to functionally associated pimips and tlie like. Generally
speaking, at probe-end 134 of cell manipulation probe 132 the ends of individual cell
manipulation elements 210 make up a matrix 145 of respective open trapping-ends 140
and have dimensions determined by the uses and size of cells to be manipulated, which
are generally significantly smaller in the micron scale. Figure 6B. For example, the size

25 of open trapping ends for the manipulation of cells is in the order of between 10'* to 10^
microns,. Therefore a typical cell-manipulation element 210 and a typical cell
manipulation probe 132 are both of a gradually decreasing dimension from butt-end 136
towards probe-end 134, as depicted in Figures 8.

30 Observation component

Although the device of the present invention can be used to manipulate cells
without actually obser\'ing any individual cell manipulated, it is generally preferable to

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observe the cells. Observation is useful, for example, for selecting cells for
manipulation, for morphological study, for direct observation of biological processes
and to confimi that desired manipulations are performed as desired. It is therefore
preferable that a device 130 of the present invention be functionally provided with an

5 observation component 150, as depicted in Figure 6A.

Many different types of observation components can be usefully coupled to a
device of the present invention, but preferably an observation component is an optical
inspection device. Optical inspection devices such as optical or CCD microscopes
having a magnification appropriate for viewing living cells in sizes in the order of 10"'

10 microns are known and are commercially available.

As is known to one skilled in the art, an optical or CCD microscope is most
effective for the morphological and morphometric characterization of a studied object.
It is therefore advantageous to provide a device of the present invention, such as device
130, with an observation component suited for the detection of fluorescence. It is known

15 to one skilled in the art that the measurement of vital fluorescence parameters
(fluorescence intensity, polarization, energy transfer) provides important information
about a living cell.

Central Control Unit

20 To allow performance of complex experiments it is preferred that a device of the

present invention such as 130 be provided with a central control unit 144 as depicted in
Figure 6A. A typical central control unit such as 144 receives commands from a user
through a user input interface 146 and activates pumps 142, observation component 150
and other components as necessary. As is clear to one skilled in the art, a central control

25 unit 144 can advantageously be implemented using a programmable computer.

As is discussed herein, central control unit 144 is also used to determine the
exact sequence of activation of pxraips 142 and other components of a device 130 of the
present invention to trap, move, observe and otherwise manipulate cells using a
cell-manipulation probe 132.

30 In a preferred embodiment, a central control unit 144 is used also to store a

record of the experiments and conditions to which each cell was exposed, vide infra.

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Although identification, examination and detennination of various states of a
cell can be performed by a human operator looking at video output produced by an
observation component of a device of the present invention, it is preferred that some or
all of these functions be performed by a central control unit. Automated image analysis
5 useful in identifying locations of cells and various morphologies is well-known and can
be implemented in the context of a device of the present invention using a central
control unit by one skilled in the art of image analysis.

Many other functions required from or possible with a central control unit are
known or obvious and will not be listed or discussed herein.

Construction of a cell manipulation device

There are many technologies available allowing one skilled in the art to
construct a cell manipulation probe of the present invention.

One preferred method for the construction of a cell-manipulation probe of the

15 present invention is free-form manufacturing (FFM) using ceramic powders. As is
known to one skilled in the art, in jfree-form manufacturing a part, such as a
cell-manipulation probe, is built "from the ground up" by sequentially dispensing layers
of ceramic powder one on top of the othCT, see for example, U.S. 6,376,148; U.S.
6,238,614; U.S. 6,228,437; U.S. 6,066,285; U.S. 6,117,612; U.S. 6,046,426; U.S.

20 5,059,266; U.S. 5,204,055 or U.S. 6,206,672. A review of the state of the art of FFM
can be found in U.S. 6,376,148, which is incorporated herein by reference for all
purposes as if fully set forth herein. It is important to note that when a ceramic powder
is used it is generally preferable to use a ceramic powder with as small a diameter as
possible, for example 10 nanometer diameter ceramic powders. Since the use of

25 jfree-form manufacture methods to construct a complex structure such as a
cell-manipulation probe of the present invention is known to one skilled in the art, this
is not further discussed herein.

A cell manipulation probe, such as cell manipulation probe 132 can be
purchased from commercial suppliers, for example from TEGS Ltd. (Saratov, Russia).

30 A preferred method for the construction of a cell-manipulation probe, such as

cell manipulation probe 132, is through the bundling of a multiplicity of glass tubes and
rods. Generally speaking, the preferred method involves three steps. In a first step, glass

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rods and tubes are bundled together in an appropriate way to assemble an incipient
cell-manipulation element or, preferably, an incipient cell-manipulation probe. The
diameter of the tubes and rods is chosen so that subsequent attachment of pumps and the
like to the butt end of the cell-manipulation probe is simple. In a second step, glass rods
5 are fused together. In a third step, one end of the bxmdle of glass rods and tubes is. drawn
in the usual manner known to one skilled in the art of glass-working so as to form the
probe-end of the cell manipulation probe. In such a way, the size of the probe-end of the
cell-manipulation probe is reduced without blocking channels and conduits, producing a
cell-manipulation probe as depicted in Figure 8A.

10 The method of use of glass tubes and rods in constructing an individual

cell-manipulation element is described in further detail with reference to Figures 9.
Extension of the method of arranging glass tubes and rods to the construction of a
cell-manipulation probe involves the construction of a multiplicity of cell-manipulation
elements and is clear to one skilled in the art upon study of the disclosure herein.

1 5 Figure 9 A is a top view of a bundle of seven glass rods 212a, 212b, 212c, 212d,

212e, 212f and 212g used in the assembly of a cell manipulation element whereas
Figure 9B is a vertical cross section of the bundle through the plane A-A. The bundle of
seven glass rods 212 can be considered to be an incipient cell-manipulation element
213. It is seen in Figure 9B that the end of central glass rod, 212g is lower than the end

20 of glass rods 212e and 212c. In fact, the ends of all six glass rods 212a througli 212f are
at the same height. The volume in incipient cell manipulation element 213 defined by
the sides of glass rods 212a through 212f and the top end of recessed glass rod 212g
corresponds to the trapping enclosiire of the ultimately fashioned cell manipulation
element. The top end of recessed glass rod 212g corresponds to the cell stop of the

25 ultimately fashioned cell-manipulation element. The interstitial volumes between seven
glass rods 212 correspond to six fluid passages that become the conduit or conduits in
the ultimately fashioned cell-manipulation element.

The use of seven glass rods 212 as depicted in Figures 9A and 9B and as
described hereinabove allows the construction of a cell-manipulation element such as

30 176 of Figure 7B, 188 of Figure 7E, 198 of 7G and 206 of 71, although for the
construction of cell manipulation elements such as 198, a glass rod having a stainless
steel wire through the central axis may replace glass rod 212g. It is important to note

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that generally the differentiation between a single conduit cell-maniptilation element
such as 176 and a multiple conduit cell-manipulation element such as 188, 198 or 206 is
not necessarily apparent in the incipient cell-manipulation element 213. Rather, after the
incipient cell-manipulation element (or incipient cell-manipulation probe) is drawn, the

5 exact nature of the ultimately fashioned cell-manipulation element is determined by
how functionally associated conduit pumps are attached to the passages. If all six
passages are connected to a single conduit pump, then a cell-manipulation element
having one conduit such as 188 is formed. If two adjacent liquid passages are connected
to one conduit pump, whereas the opposing pair of liquid passages is connected to a

10 second conduit pump then a cell manipulation element having two peripheral conduits
such as 188, 198 or 206 is formed.

In Figure 9C a method of construction for a cell manipulation elements such as
182 of Figure 7D, 202 of 7H or 206 of 71 is depicted. Analogously to the method
described immediately hereinabove and depicted in Figure 9A and Figure 9B, six glass

15 rods 212a, 212b, 212c, 212d, 212e and 212f are arrayed about a glass tube 214. Since
the end of glass tube 214 is lower than the ends of glass rods 212, the volume defined
by the sides of glass rods 212a through 212f and the top end of recessed glass tube 214
corresponds to the trapping enclosure of the ultimately fashioned cell manipulation
element. The top end of recessed glass tube 214 corresponds to the cell stop of the

20 ultimately fashioned cell-manipulation element. The interstitial volumes between six
glass rods 212 and recessed glass tube 214 correspond to six passages that potentially
become conduits in the ultimately fashioned cell-manipulation element. The hollow
bore of recessed glass tube 214 corresponds to the axial conduit in the ultimately
fashioned cell-manipulation element. To fashion a cell wall penetrating tool 204 of a

25 cell manipulation element 202, the end of recessed glass tube 214 is sharpened.

It is clear to one skilled in the art upon studying the description herein, that in
order to fashion an incipient cell-manipulation probe instead of an incipient cell
manipulation probe, more than seven glass rods and tubes are bundled together. It is
also clear to one skilled in the art upon reading the description herein that other types of

30 cell manipulating elements or other elements for constructing a cell manipulating probe
such as a waste element or an isolation element can be produced in an analogous
fashion to that described hereinabove.

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As stated above, once chosen the glass rods and tubes making up an incipient
cell-manipulation element or incipient cell manipulation probe are bundled together in
an appropriate fashion, the rods and tubes are fused together. Thereafter, the appropriate
end of the incipient cell-manipulation element or incipient cell manipulation probe is
drawn out into the shape depicted in Figure 8A to make a cell manipulation element or
cell manipulation probe, respectively.

In an alternative embodiment, modular cell manipulation elements are made and
assembled to make a cell manipulation probe. In Figures 10 are depicted examples of
modular cell manipulation elements.

In Figure lOA is depicted one embodiment of a simple modular cell
manipulation element 216 with two conduits, 218 and 220. The horizontal cross-section
of cell manipulation element 216 is substantially square. It is important to note that
other embodiments of similar cell manipulation elements potentially have non-square
cross-sections. Conduits 218 and 220 function analogously to conduits described
hereinabove. Illustrative but non-limiting dimension of cell manipulation element 216
are a height of 1000 micron, a width of 20 micron and a depth of 20 micron. The top of
cell manipulation element 216 is recessed, creating a well-like enclosure, 222 serving as
a dimple or trapping enclosure as described hereinabove. An illustrative but
non-limiting depth of well-like enclosure 222 is 10 micron. Other types of elements,
especially cell manipulation elements, can be conceived for use together with modular
cell manipulation elements such as 216. Examples of such other types of elements
include a modular cell manipulation element 224 with three conduits 218, 220, and 226,
depicted in Figure lOB and a modular cell manipulation element 228 with two conduits
218 and 220 and a cell wall penetrating tool 230, depicted in Figure IOC.

Assembly of a cell manipulation probe jfrom modular cell manipulation elements
such as 216, 224 and 228 is done by providing an assembly plate 232, depicted in
Figure lOD. Assembly plate 232 is a monolithic structure having a multiplicity of pin
connector triplets 234, which fit into the bottom openings of conduits 218, 220 and 226
or a conduit of cell wall piecing tool 230 of modular cell manipulation elements 216,
224 and 228. At the bottom of assembly plate 224, each pin connector 226 is connected
to fluid lines (not depicted), which connect to functionally associated conduit pumps
(not depicted). Thus a multiplicity of modular cell manipulation elements 216 can be

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disposed in exact juxtaposition on assembly plate 224, thus providing functionally
associated conduit pumps to cause fluid to flow through conduits 218 and 220.

The various components necessary for implementing a cell manipulation probe
using modular cell manipulation elements, including modular cell manipulation
5 elements can be purchased from commercial suppliers, for example from TEGS Ltd.
(Saratov, Russia).

Once a cell-manipulation probe is constructed, the assembly of a device of the
present invention from commercially available conlponents is well within the ability of
one skilled in the art upon study of the description herein.
10 Suitable computers, software and related peripheral equipment for implementing

a central control unit are available from International Business Machines Corporation
(Armonk, New York, USA). Any required unique or dedicated software can be written
by one skilled in the art of computer programming.

Suitable CCD microscopes for implementation of an observation component are
15 available, for example, from Hirox Co Ltd. (Suginami-Ku, Tokyo, Japan).

Flow-generators and peripheral equipment suitable for implementing the present
invention are available from, for example, GeSIM (Grosserkmannsdorf, Germany) or
Fluidigm Corporation (San Francisco, California, USA).

20 Steps used in building experiments

A device such as 130 depicted in Figures 6 designed for manipulating individual
cells found in a liquid medium (especially a cell culture medium) and allows
performance of many novel and useful experiments. Experiments are done by
perfoming a series of one or more steps (also traned manipulations or functions). A few

25 standard steps that are useful building blocks for various experiments are described in
djstail hereinbelow with reference to device 130 as depicted in Figures 6 and especially
with reference to Figure 6B. Unless otherwise stated, for the examples below it is
assumed that all cell manipulation elements are similar to cell manipulation element 182
depicted in Figure 7D with a reagent inlet system such as depicted in Figure 7F,

30 Useful steps, manipulations and functions include loading and isolating of a cell

in a trapping enclosure, washing a cell, suspending a cell for observation, penetrating a
cell wall {e.g, for remo\ang a sample or inoculation with a reagent), exposure of a cell to

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a reagent, allowing a cell to intCTact with other cells, select a cell, sort cells and separate
types of cells from others.

Cell Loading and Isolating

Generally but not necessarily, a first step in an experiment is the loading of cells
into trapping enclosures 140 of device 130.

Sample inlet pump 158 receives a command from central control unit 144 to
operate, loading a sample of cell-containing liquid through sample inlet 152 onto matrix
145 on probe-end 134 of cell manipulation probe 132, Figure 6C. In an alternate
embodiment using the variant of cell manipulation probe 145 depicted in Figure 6D, a
cell-containing sample is introduced through sample introduction element 163 and
flows out onto matrix 145.

For all trapping enclosures 140 in which it is desired to load cells 142, a
functionally associated first conduit pump 190 and a second conduit pump 192 are set
by central control device 144 to suction mode. A cell 142 in proximity of a respective
open trapping-end 170 is drawn into a respective trapping enclosure 140 and is drawn
into a trapping enclosure 140 until making contact with a respective cell-stop 184.

After a certain period of time when it is estimated that cells are trapped in all
desired trapping enclosures 140, associated first conduit pumps 190 and second conduit
pumps 192 are set to inactive mode by central control device 144. Observation
component 150 receives a command from central control element 144 to examine all
trapping enclosures 140 to ascertain if cells 142 are trapped therein. First conduit pump
190 and second conduit pump 192 of trapping enclosures 140 that are empty are again
set to suction mode until all desired trapping enclosures 140 hold cells 142.

Once cells 142 are held in all desired trapping enclosures 140, a waste pimip
functionally associated with a waste element is set to suction mode by central control
unit 144. Excess cells and liquids not trapped in trapping enclosures 140 are removed
and disposed of through inlet 148 of the waste element. Subsequently, fluid inlet pump
160 is activated to dispense an appropriate cell culture medium through fluid inlet 154
onto matrix 145 by central control unit 144.

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In one embodiment, it is desired that cells 142 be trapped in substantially all
trapping enclosures 140. In such an embodiment, first conduit pumps 190 and second
conduit piimps 192 associated with all trapping enclosures 140 are set to suction mode
and consequently draw cells 142 into a respective trapping enclosure 140.

In a different embodiment, it is desired to maintain a maximal flexibility for
movement of cells 142. In such an embodiment, in a first step first conduit pumps 190
and second conduit pumps 192 associated with only non-neighboring trapping
enclosures 140 are set to suction mode. In subsequent steps cells can be moved and
transferred between trapping enclosures with great ease as every cell 142 has a free path
to every trapping enclosure 140 of matrix 145, vide infra.

Cell Observation

In one embodiment of the present invention, a cell 142 in a trapping enclosure
140 is observed through a respective open trapping-end 170. In another embodiment,
the walls of a trapping enclosure 140 are transparent and a cell 142 held therein is
observed therethrough.

In a preferred embodiment, when cell manipulation elements are provided with a
conduit or trapping enclosure deep enough to allow deep cell entry, cells 142 not to be
observed are drawn inside the cell manipulation element so that only one or only a few
cells 142, each in a trapping enclosure 140, remain in the proximity of a respective open
trapping-end. Observation is then performed only on those cells 142 in proximity of a
respective open trapping-end 170.

Most preferably, if a cell 142 is held in a trapping enclosure 140 of a cell
manipulation element suitable for suspending cell 142 above the trapping enclosure
using the Venturi effect (such as cell manipulation element 182), as described above,
then cell 142 to be observed is suspended for observation. Central control unit 144 sets
a conduit pump functionally associated with axial conduit 186 to injection mode while
conduit pumps functionally associated with one or more peripheral conduits 174 are set
to suction mode. As discussed hereinabove, cell 142 trapped in trapping enclosure 140
is elevated out of trapping enclosure 140 and suspended thereabove. The advantages of
suspending a cell 142 to be observed in such a way are manifold. Cell 142 can be
selectively and intensely illuminated using incoherent and/or coherent hght aimed at

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cell 142. Light scattering and shadows are eliminated. Importantly the greater intensity
of light allows the use of a small camera aperture, increasing depth of field and leading
to the recording of a sharper image. Further, the intensity of lighting can be chosen to
maximize contrast and thus maximize the observable details.
5 After cell 142 is examined, associated first conduit pump 190 and second

conduit pump 192 may be both set to suction mode to retum cell 142 to trapping
enclosure 140. Thereafter an additional cell can be examined if required.

Reagent addition through vial reagent inlet

10 One way to expose cells to reagents is through a vial reagent inlet 156. Central

control unit 144 activates vial reagent inlet pump 162 to dispense an amount of a
reagent through a vial reagent inlet 156 onto matrix 145. However, in order for a cell
142 held in a trapping enclosxire 140 to come in contact with a thus-dispensed reagent,
the reagent must be brought into trapping enclosure 140.

15 If all cells in all trapping enclosures are to be exposed to the reagent (for

example, in the case where the reagent is a nutrient or a fluorescent marker), then one or
both conduit pumps 190 and 192 associated with all trapping enclosures 140 are set to
suction mode, drawing an amount of reagent into each trapping enclosure 140.

If, however, it is desired that only certain cells 142 be exposed to a given

20 reagent, then one or both conduit pumps 190 and 192 functionally associated only with
the respective trapping enclosures 140 of certain cells 142 are set to suction mode.

Reagent addition through a cell-manipulation component

As stated above, the fact that a given trapping enclosure 140 is fiinctionally

25 associated with two independently controllable conduit pumps 190 and 192 allows
"washing*' of trapping enclosure 140 or of a cell 142 held within trapping enclosure 140
by using one fimctionally associated conduit pump to inject a washing liquid
simultaneously with extraction of the washing liquid using the other conduit pump.

As stated above, the fact that a trapping enclosure 140 is fimctionally associated

30 with two independently controlled conduit pumps 190 and 192 together with a reagent
injection pump 194 as depicted in Figure 7F allows selective exposure of a cell 142 in
trapping enclosure 140 to a reagent. Reagent injection pump 194 is set to add a desired

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amount of reagent to the flow produced by first conduit pump 190 is set to injection
mode. The flow of fluid from conduit 174 carries reagent to trapping enclosure 140,
thus exposing cell 142 to the reagent. To prevent leakage of the reagent out of trapping
enclosure 140, second conduit pump 192 is set to suction mode, actively removing
5 reagent and other fluids from enclosxire 140.

Moving a cell to a different trapping enclosure

The movement of a cell 142 held in a trapping enclosure 140 to another trapping
enclosure by a series of transfers between neighboring trapping enclosures is performed
10 according to the method of the present invention and is clear from the description
hereinabove. Such a transfer is schematically depicted in Figures 11.

A cell 142 is held in a trapping enclosure of cell manipulation element 236,
Figure 11 A.

As depicted in Figure 11 A, conduit pumps ftmctionally associated with cell
15 manipulation element 236 are set to injection mode and conduit pumps functionally
associated with neighboring cell manipulation element 238 are set to suction mode. A
flow of liquid is produced from ceil manipulation element 236 to neighboring cell
manipulation element 238, depicted as arrows 240.

As depicted in Figure IIB, the force produced by flow of liquid 240 ejects cell
20 142 from cell manipulation element 236 and carries cell 142 towards neighboring cell
manipulation element 238.

As depicted in Figure IIC, cell 142 is ultimately carried into the trapping
enclosure of neighboring cell manipulation element 238.

A cell is moved from any trapping enclosure to any other unoccupied trapping
25 enclosure along a path defined by a series of unoccupied neighboring enclosure,
according to the method of the present invention and as described herein and in Figure
3. The ability to move an individual cell from one trapping enclosure to any other
trapping enclosure allows the performance of many useful experiments.

For example, it may be desirous to expose similar cells to a series of reagents,
30 but to compare the effect of changing the order of exposure or the length of time a cell
is exposed to a given reagent. To perform such an experiment, cells are loaded into

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trapping enclosures. The cells are sequentially transported to different trapping
enclosures provided with specific reagents and therein exposed to reagents, as required.

It is also often desired to study the interaction of two different types of cells.
Cells of a first type are loaded into trapping enclosures, preferably unoccupied trapping
5 enclosures. Cells of a second type are loaded into other trapping enclosxires, preferably
unoccupied trapping enclosures. A cell of each type is moved, as described above, to a
multi-cell reaction enclosure 151. In another embodiment contingent on a properly sized
trapping enclosure, a first cell is moved into a trapping enclosure already occupied by a
second cell. Observation component 150 is used to observe any interaction. When
10 desired, the two cells are ejected fi*om multi-cell reaction enclosure 151 and another pair
of cells is loaded therein for study.

It is also often desired to move a specific cell or cells held in a trapping
enclosure to an isolation element in order to collect cells of a certain type for fiirther
study. Each individual cell is transferred through a series of trapping enclosures, firom
15 one trapping enclosure to a neighboring trapping enclosure, until cell is located in a
trapping enclosure 140 neighboring an isolation inlet 146 of an isolation element. The
cell is ejected firom trapping enclosure 140 while a conduit pump functionally
associated with the isolation element is activated so as to draw cell 142 into the isolation
element through isolation inlet 146.

20 Analogously, it may also be desired to discard a specific cell 142 firom a

trapping enclosure. Cell 142 is transferred firom one trapping enclosure 140 to a
neighboring trapping enclosure 140 until cell 142 is located in the vicinity of waste inlet
148. Cell 142 is ejected fi-om trapping enclosure 140 while a pump fimctionally
associated with the waste element is activated so as to draw cell 142 through waste inlet

25 148.

In most embodiments of the present invention, a device 130 is configured to
allow simultaneous activation of many pumps and thus perform manipulations and
fimctions simultaneously. Thus, in one embodiment of the present invention, numerous
cells are individually manipulated simultaneously using one device of the .present
30 invention such as 130. Thus, in another embodiment of the present invention, numerous
cell-cell interactions are simultaneously performed using one device of the present
invention such as 130. In another embodiment, nxmierous specific treatments and

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manipulations of numerous individual cells are simultaneously performed using one
device of the present invention such as 130. In another embodiment, many cells are
simultaneously moved from a first trapping enclosure to another trapping enclosure, as
desired. The preparation of an algorithm that calculates an optimal series of steps, where

5 in each step one or more cells 142 are transferred from one trapping enclosure 140 to
another, is well within the ability of one skilled in the art of computer programming.
Such an algorithm is easily implemented in central control unit 144 of a device 130 of
the present invention. Once an optimal smes of steps is found, central control unit 144
sets the various conduit pumps to perform the appropriate actions according to the

10 calculated series of steps so as to relocate and rearrange cells 142 as required.

Preferably, at any given moment, the locations of all cells 142 in trapping
enclosures 140 of a cell manipxjlation probe 132 are recorded in central control unit 144,
Preferably, central control unit 144 also keeps a record of the manipulations that any
given cell has imdergone. In such a way, data can be easily analyzed.

Exemplarv Experiments

One skilled in the art, upon perusal of the description of the present invention
hereinabove is able to construct a device of the present invention and to plan a plethora
of heretofore-impossible experiments, manipulations and studies that yield hitherto

20 unavailable scientific information. For sunplicity, the words experiment, manipulation
and study will be used hereinbelow interchangeably. The manipulation of individual
cells enabled by the teachings of the present invention provides far more information for
the understanding of biological implications of phenomena under study compared to
bulk studies (eg., using cuvettes or microplates), flow-through cytometry studies or

25 static cytometry studies.

According to a preferred embodiment of the present invention, specific cells are
selected for real-time high-throughput study. It is important to note that some of the
studies performed using the teachings of the present invention including some of those
described hereinbelow may have been previously manually performed. It is important to

30 note, however, that the teachings of the present invention are the first that allow the
perfomiance of such studies automatically on many cells simultaneously as opposed to
manually by highly skilled individuals.

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An additional advantage of the present invention is that selected cells can be
maintained for a relatively long period of time in varions environments, allowing a
single cell to be used for many different experiments. For instance, using a device of the
present invention a large number of cells is exposed to a stimulus and cells that respond

5 to the stimulus in a certain way are isolated, for example, in trapping enclosures of a
cell manipulation probe or by transfer to an isolation element as described above.
Thereafter, the reaction of the isolated cells to other stimuli can be studied. In summary,
once a cell having xmusual or otherwise interesting properties is identified the teachings
of the present invention are used to isolate and further manipulate the cell, thereby

10 pinpointing an investigation to a subgroup of relevant cells, reducing investigation time
and saving valuable resources. Additionally, since such an investigation is done on a
cell-by-cell basis, better and more precise data is obtainable, for example, for increased
diagnostic accuracy.

The teachings of the present invention can also be used for sorting cells or

15 selecting specific cells firom a large population of cells. Such procedures include the
selective removal of undesired cells or the harvesting of desired cells found in small
proportions from amongst other cells, activities that cannot be performed using methods
known in the art. Single-cell therapy can be performed by identifying and isolating only
cells exhibiting a pathology from amongst a group of many cells, treating the isolated

20 cell in some fashion (e.g"., exposure to a reagent) £Uid returning the thus-treated cell to
the original group. Groups of cells having exceptional interest include, for example,
lymphocytes in blood, T jurkat cell lines, lymph node cells, tumor cells and other
representative groups.

When applied to viable cells taken from a living organism (such as a mammal,

25 both human and non-human), the teachings of the present invention enable the treatment
of biological fluids from living organisms (such as a manraial, both human and
non-human) by procedures including but not limited to the manipulation of individual
cells or removal of abnormal cells (especially harmful abnormal cells) from amongst
normal cells. Such a treatment can be considered a form of selective manipulation of

30 particles (especially cells) in a biological fluid, including but not limited to lymphatic
fluids, blood, cerebral spinal fluids, semen, saliva, synovial fluid, bone marrow,
cochlear fluid, fluid extracted from tumors, especially malignant tumors, ovarian fluid.

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amniotic fluid and chorionic fluid. In one embodiment, such treatments can be
perfonned "off-line", that is by removing a sample of a biological fluid from an
organism (both human and non-human) treating {e.g, selecting or manipulating cells)
the fluid in a device of the present invention and returning the treated fluid to the
organism. In a preferred embodiment, such treatments are performed "on-line", that is
by directly attaching an input line from the organism to a device of the present
invention, and returning the treated fluid to the organism. An "on-line" treatment
according to the present invention of an organism is depicted in Figure 12, where an
input line 243 is attached from an organism 245 to a device of the present invention
247. In device 247 a desired treatment is performed and the treated fluid is returned
from device 247 through an output line 249 back to organism 245. In such an
embodiment it is advantageous that the central control unit of device 247 be configured
to automatically detect and treat cells, for example by using automated image analysis.

Another experiment that is performed using the teachings of the present
invention involves loading cells to be studied into trapping enclosures of
cell-manipulation elements equipped with hollow penetrating-tools. The cell walls are
penetrated as described hereinabove and the penetrated cell inoculated with a first
reagent injected through the conduit in the hollow-penetrating tool. The thus-prepared
cell is exposed to a second reagent in a manner as discussed hereinabove. The reaction
of the inoculated cell is then observed.

Another related experiment involves the improved fertilization of ova (an
improved form of IVF). A fluid sample containing viable ova is loaded onto a cell
manipulation probe of the present invention. The ova are stored in trapping enclosures
equipped with hollow penetrating tools. The walls of the ova are penetrated and sperm
is injected therethrough into the ova. Each individual ovum is released firom the
penetrating tool but retained inside a trapping enclosure. Cell division and development
is observed and only ova that are successfully fertilized and apparently viable are
moved to an isolation element for transfer to a uterus.

A device of the present invention can also be used to transfer cells to a different
device, for example a biochip processor as described in PCT patent application
PCT2001 / IL 0000992 published as WO 03/035824. Cells are loaded and undergo
manipulation as described above including selection, sorting and exposure to reagents

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and other stimuli. When it is so desired, cell-manipulation probe 132 is brought in
contact, in proximity or somehow physically connected with a fluid passage (not
depicted) with the surface of biocMp processor 242 (or the like such as a microwell
plate such as a Nimc 384-well plate) as depicted in Figure ISA. A specific cell or cells
5 are deposited from chosen trapping enclosures 244 and 246 in cell-manipulation probe
132 to identifiable locations 248 and 250 on biochip processor 242. Biochip processor
242 is then used to handle the selected cells in the usual way and as described in WO
03/035824. In such a way, selected cells identified and isolated using the teachings of
the present invention may be grown and allowed to proliferate. In another embodiment

10 of the invention, this process is reversed so as to load a cell-manipulation probe 132 of
the present invention with cells from a biochip processor 242 (or the like such as a
microwell plate such as a Nunc 384-well plate). Cell manipulation probe 132 is brought
in proximity to the surface of biochip processor 242, Figure 13B. Associated conduit
pumps are set to suction mode, loading cells 142 from biochip processor 242 to trapping

15 enclosures of cell manipulation probe 132. The ability to easily couple two advanced
research methods exponentially increases the significant results gleaned from an
experiment. Such coupling is exceptionally advantageous when the two research
methods are format compatible, that is, when the number, and preferably geometric
arrangement, of trapping enclosures in cell manipulation probe 132 are substantially

20 similar or even identical to the identifiable locations (such as 248 and 250) of biochip
processor 242.

It is appreciated that certain features of the invention, which are, for clarity,
described in the context of separate embodiments, may also be provided in combination
in a single embodiment. Conversely, various features of the invention, which are, for
25 brevity, described in the context of a single embodiment, may also be provided
separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific
embodiments thereof, it is evident that many alternatives, modifications and variations
will be apparent to those skilled in the art. Accordingly, it is intended to embrace all
30 such alternatives, modifications and variations that fall within the spirit and broad scope

pCt/IL2004/000l94

46 f

for the sake ot

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WHAT IS CLAIMED IS:

1. A method of moving an individual small object from an initial location

to a destination location comprising:

a. defining a series of N+1 locations Lo-Ln, N being an integer greater
than 1 wherein U is the initial location and Ln is the destination location; and

b. for i from 0 to N-1, successively moving the individual small object
from a location Li to a location Lj+i

wherein said moving is effected by applying an attractive force from said location Li+i
to the individual small object so as to cause the individual small object to move from a
said location Li to a said location Ln-i..

2. The method of claim 1 wherein said location Li is fixed relative to said
location Li+i.

3. The method of claim 1 further comprising applying a repulsive force to
the individual small object from location Li-

4. The method of claim 3 wherein said repulsive force is selected from the
group consisting of physical, electrical and magnetic forces.

5. The method of claim 3 wherein said repulsive force is a resuU of a flow
of fluid from said location Li.

6. The method of claim 5 wherein said flow of fluid is generated by
injection of a fluid from the vicinity of location U-

7. The method of claim 1 wherein said attractive force is selected from the
group consisting of physical, electiical and magnetic forces.

8. The method of claim 1 whereijo'said attaractive force is a result of a flow
of fluid towards said location Lj+i .

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9. The method of claim 8 wherein said flow of fluid is generated by suction
of a fluid from the vicinity of location Li+i.

10. The method of claim 1 wherein at least one of said locations Lo...Ln,
comprises an enclosure of a size sufficient to substantially enclose the individual small
object.

11. A method of moving an individual small object in a fluid from a first
location to a second location comprising:

a. drawing fluid towards the first location so as to produce a first force localizing
the small object at the first location; and

b. subsequenfly drawing fluid towards the second location so as to produce a
second force localizing the small object at the second location.

12. The method of claim 11 wherein said first location is fixed relative to
said second location,

13. The method of claim 11 wherein said drawing of fluid towards the first
location and towards the second location is performed by suction of the fluid by a flow
generator.

14. The method of claim 11 further comprising reducing the magnitude of
said first force so as to assist in said localizing of the small object at the second location.

15- The method of claim 1 1 fiirther comprising eliminating said first force so
as to assist in said localizing of the small object at the second location.

16. The method of claim 1 1 further comprising injecting fluid from the first
location so as to produce a repulsive force to assist in said localizing of the small object
at the second location.

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17. The method of claim 11 wherein said first location and said second
location comprise enclosures of a size sufficient to substantially enclose the individual
small object.

18. The method of claim 17 wherein said drawing of fluid towards the first
location and towards the second location is performed by suction of said fluids from
within a respective said enclosure.

19. The method of claim 1 or 11 wherein the order of the size of the
individual small object is equal to or less than about 10^ micron.

20. The method of claim 1 or 11 wherein the order of the size of the
individual small object is equal to less than about 10^ micron.

21. The method of claim 1 or 11 wherein the order of the size of the
individual small object is equal to less than about 10^ micron.

22. The method of claim 1 or 11 wherein the order of the size of the
individual small object is equal to less than about 10^ micron.

23. The method of claim 1 or 11 wherein the individual small object is
selected firom the group consisting of cells, crystals, bacteria, viruses, proteins,
polymers, macromolecules, ions and atoms.

24. The method of claim 1 or 1 1 wherein the small object is a cell.

25. The method of claim 1 or 1 1 wherein said fluid is a liquid.

26. The method of claim 1 or 11 wherein said liquid is a cell culture
medium.

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27. A cell manipulation device comprising:

a. at least N locations, each one of said N locations having a fixed position
relative to at least one other of said N locations; and *

b. at least two flow generators, each flow generator being functionally associated
with a said location

wherein N is at least two,

wherein at least two of said flow generators are independently settable to a mode
selected from the group of modes including a suction mode and an inactive mode
and wherein at least two of said independently settable flow generators are each
associated with a different said location.

28. The cell manipulation device of claim 27 wherein N is at least three and
wherein at least three of said at least N locations are arranged in a 1 dimensional array.

29. The cell manipulation device of claim 27 wherein N is at least four and
wherein at least four of said at least four locations are arranged in a 2 dimensional array.

30. The cell manipulation device of claim 27 wherein N is at least five and
wherein at least five of said at least five locations are arranged in a rectangular lattice
aixay.

3 1 . The cell manipulation device of claim 27 wherein N is at least seven and
wherein at least seven of said at least seven locations are arranged in a hexagonal lattice
array.

32. The cell manipulation device of claim 27 wherein N is at least ten.

33. The cell manipulation device of claim 27 wherein N is at least 19.

34. The cell manipulation device of claim 27 wherein N is at least 24.

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35. The cell manipulation device of claim 27 wherein N is at least 36.

36. The cell manipulation element of claim 27 wherein at least one of said
flow generators is independently settable to a mode selected from the group of modes
including a suction mode, an injection mode and an inactive mode.

37. The cell manipulation element of claim 27 wherein each one of said flow
generators is independently settable to a mode selected from the group of modes
including a suction mode and an inactive mode

38. The cell manipulation device of claim 27 wherein at least two of said at
least N locations are substantially defined by the presence of an inlet of a respective said
flow-generator.

39. The cell manipulation device of claim 38 wherein said at least two of
said at least N locations are substantially depressions in a surface.

40. The cell manipulation device of claim 27 wherein at least two of said at
least N locations substantially comprise open trapping-ends of enclosvires.

41. The cell manipulation device of claim 40 wherein emerging within at
least two of said at least two enclosures is an inlet of a respective flow-generator.

42. The cell manipulation element of claim 40 wherein the order of the size
of said at least two open trapping-ends is equal to or less than about 10^ micron.

43. The cell manipulation element of claim 40 wherein the order of the size
of said at least two open trapping-ends is equal to or less than about 10^ micron.

44. The cell manipulation element of claim 40 wherein the order of the size
of said at least two open trapping-ends is equal to or less than about lO' micron.

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45. The cell manipulation element of claim 40 wherein the order of the size
of said at least two open trapping-ends is equal to or less than about 10^ micron.

46. The cell manipulation element of claim 40 wherein at least one of said N
locations is associated with at least two flow generators and wherein each one of said at
least two flow generators is independently settable to a mode selected from the group of
modes including a suction mode and an inactive mode.

47. The cell manipulation element of claim 40 wherein at least one of said N
locations is associated with at least three flow generators and wherein each one of said
at least three flow generators is independently settable to a mode selected from the
group of modes including a suction mode and an inactive mode.

48. The cell manipulation device of claim 27 wherein any one said location
is no more than 1000 micron distant from at least one other said location.

49. The cell manipulation device of claim 27 wherein any one said location
is no more than 100 micron distant from at least one other said location.

50. The cell manipulation device of claim 27 wherein any one said location
is no more than 10 micron distant from at least one other said location.

5 1 . The cell manipulation device of claim 27 wherein any one said location
is no more than 1 micron distant from at least one other said location.

52. The cell manipulation device of claim 27 wherein at least one of said N
locations is configured to suspend a cell at distance from said at least one of said N
locations using a Venturi effect.

53. The cell manipulation device of claim 27 wherein at least one of said N
locations is provided with a cell wall penetrating tool.

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54. The cell manipulation device of claim 53 wherein each said cell wall
penetrating tool provided with a said location comprises a member pointing in a
direction substantially opposite a flow generated by a said flow generator functionally
associated with said location when set to suction mode.

55. A method of studying a cell comprising suspending said cell individually
in a liquid by using a Venturi effect.

56. A device for suspending a cell in a liquid comprising:

a. a substantially hollow body having an open trapping-end and a central

axis;

b. emerging within said hollow body, at least one outlet of a flow
generator configured to inject liquid into said hollow body and through said
open trapping-end in a first flow substantially parallel to said central axis of said
hollow body; and

c. emerging within said hollow body, at least one inlet of a flow
generator configured to draw liquid through said open trapping-end and out of
s£iid hollow body in a second flow substantially parallel to said central axis of
said hollow body;

wherein said first flow is closer to said central axis than said second flow.

57. A method of penetrating a cell wall comprising:

a. immersing, in a liquid, a cell wall penetrating tool pointing in a first
direction; and

b. applying suction from a second direction opposite said first direction
so as to cause a cell in said liquid to be carried against said cell wall penetrating
tool

v/here the intensity of said carrying is sufficient to cause a wall of said cell to be
penetrated.

58. A device for penetrating a cell wall comprising:

a. a substantially hollow body having an open trapping-end;

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b. emerging within said hollow body, at least one inlet of a flow
generator configured to draw liquid out of said hollow body in a flow having a
first direction; and

c. a cell wall penetrating tool pointing in a second direction substantially
opposite said first direction.

59. A method for selecting cells comprising:

a. providing a group of cells;

b. isolating cells from said group of cells each of said cells in an
individual enclosure;

c. examining each isolated cell; and

d. selecting isolated cells fiilfiUing at least one criterion.

60. The method of claim 57 further comprising:

e. separating said selected cells firom said group of cells.

61 . A method for treating a cell-containing biological fluid comprising

a. providing the cell-containing biological fluid;

b. identi^ng cells to be treated in the biological fluid;

c. differentiating between said cells to be treated and other cells; and

d. either or both:

i. physically separating said cells to be treated firom said other cells; and /

ii. directly manipulating said cells to be treated
thus treating the biological fluid.

62. The method of claim 61 wherein during said identifying each cell is
isolated in an individual enclosure.

63. The method of claim 61 wherein said manipulating is selected from the
group consisting of destroying said cells to be treated, exposing said cells to be treated

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to a reagent, exposing said cells to be treated to radiative energy and penetrating cell
walls of said cells to be treated

64. The method of claim 61 wherein the biological liquid is selected from
the group consisting of lymphatic fluids, blood, cerebral spinal fluids, semen, saliva,
synovial fluid, bone marrow, cochlear fluid, fluid extracted from tumors, ovarian fluid,
amniotic fluid and chorionic fluid.

65. The method of claim 61 further comprising:

e. subsequently to d, directing the biological liquid into a living organism.

66. The method of claim 61 wherein said providing the biologicsd liquid,
comprises a step of taking the biological liquid from a living organism.

67. The method of claim 62 further comprising

e. subsequently to d, directing the biological liquid into a living organism
and wherein said providing the biological liquid includes a step of directing the
biological liquid to flow from said living organism to said individual enclosures.

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SUBSTITUTE SHEET (RULE 26)

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2/25

SUBSTITUTE SHEET (RULE 26)

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3/25

SUBSTITUTE SHEET (RULE 26)

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4/25

FIG.4

SUBSTITUTE SHEET (RULE 26)

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5/25

SUBSTITUTE SHEET (RULE 26)

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6/25

SUBSTITUTE SHEET (RULE 26)

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145

134

FIG.6B

SUBSTITUTE SHEET (RULE 26)

PCT/IL2004/000194

8/25

FIG.6C

SUBSTITUTE SHEET (RULE 26)

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SUBSTITUTE SHEET (RULE 26)

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10/25

166

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174

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.164

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FIG.7A

166

174

.176

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FIG.7B

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FIG,7G

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FIG.7I

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14/25

FIG.8B

SUBSTITUTE SHEET (RULE 26)

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SUBSTITUTE SHEET (RULE 26)

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216

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FIG.lOA
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224

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228

FIG.IOC

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234

FIG.IOD

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142

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Full text of "USPTO Patents Application 10561839"

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